Gibberellic acid compounds, and preparation and use thereof



July 8, 1958 P. w. BRIAN ET AL 2,842,051

GIBBERELLIC ACID COMPOUNDS, AND

PREPARATION AND USE THEREOF Filed June 27, 1955 Transmnsslon. 8 8 8 6 Transmlss'lon.

5600 $506 5406 5.206 5006 2 306 280624110 F900 mice fioo 12,00 50o l mo FIG, I, Frzqueng (cm*) Frequency (cm") IOOF Transmission.

MARGARET EDITH RADLEY PHILIP JOHN CURTIS GEORGE WYNNE ELSON o l l l n l I400 I300 I200 H00 I000 900 800 700 FIG. 2.

Fmquzncj (cm").

BY W M d W ATTORNEYS United States Patent GIBBERELLIC ACID COMPOUNDS, AND PREP- ARATION AND USE THEREOF Percy Wragg Brian, Margaret Edith Radley, Philip John Curtis, and George Wynne Elson, Welwyn, England, assignors to Imperial Chemical Industries Limited, London, England, a corporation of Great Britain Application June 27, 1955, Serial No. 518,226

Claims priority, application Great Britain June 30, 1954 11 Claims. (Cl. 71-25) This invention relates to a new organic acid having plant growth promoting properties, and to active derivatives thereof, in particular salts and esters. The invention further relates to a method of producing the acid by a metabolic process. The invention further relates to compositions for agricultural use containing the new acid or an active derivative thereof.

We have found that from a culture of Gibberella fujikuroi (Sawada) Wollenweber Fusarium mom'liforme Sheldon) there may be extracted a new organic acid having the property of promoting plant growth. This acid is hereinafter termed gibberellic acid.

The present invention therefore provides a new chem ical compound, gibberellic acid, such as is obtainable from a culture of Gibberella fujikuroi, the acid being characterised by the following properties:

Infra-red absorption curve (Nujol mull) of material crystallised from ethyl acetate-light petroleum (B. P. 60-80" C.) as shown in the accompanying drawing.

X-ray powder diffraction lines as shown in Table I. The intensities I of the lines on the powder photographs are estimated by reference to the strongest line which is assigned an intensity of 10.

2,842,051 Patented July 8, 1958 TABLE I X -ray powder dzfiractmn lines of gibberellic and (ex ethyl acetate) Spacing Intensity Spaclng Intensity 10. 20 5b 2. 786 1 s. 73 2b 2. 687 1 7. 33 5b 2. 549 2 6. 59 w 2. 435 2 0.01 5b 2. 418 w a. 31 10b 2. 3.50 1 5. 0s 35 2. 305 1 4. s4 3% 2. 249 w 4. so 7 2.112 1 4.33 3 2.120 1 4.03 3 2. 040 w 3. w 2. 003 w 3. 714 3 1.971 w 3. 591 3 1. 936 w 3. 389 1m 1. 920 w 3. 214 1 1. 863 m 3.085 to 1. 353 w 2. 993 1, 4 1. 780 w 2. 882 1% 1. 698 w w denotes a. weak reflection. b denotes a broad (lill'use reflection. The third figure beyond the decimal point is barely significant.

'iize invention also provides derivatives gibberellic acid, in particular salts and esters thereof, having plant growth-promoting properties. Examples of the esters include alkyl esters and, specifically, the methyl, ethyl, isopropyl, butyl and octyl esters. Compounds in which the alcoholic hydroxyl groups of gibberellic acid are esterified with aliphatic or aromatic acids, specifically acetic, butyric and benzoic acids, comprise an additional type of ester of gibberellic acid. The salts preferably uti lized are the alkali metal salts and other salts such as the alkaline earth salts, e. g., calcium.

The present invention also provides mixtures having plant growth-promoting properties comprising gibberellic acid, or an active derivative thereof, and a diluent. The diluent may be liquid or solid and may consist of or contain plant nutrients, particularly sources of nitrogen.

Further, the present invention provides a process of producing gibberellic acid, or mixtures containing said acid, from a culture of Gibberella fuiikuroi.

Gihberellic acid may be produced by Gibberella fujikaroi when grown as a surface cuiture or as a deep, stirred and aerated culture in a suitable aqueous medium. A suitable medium must contain a carbon source (e. g. sucrose, glucose or glycerol, at 2-30% W./v.), an animonium salt or a nitrate or a digest of protein (e. g. pep tone) as nitrogen source (to give a concentration of N in the medium of OBI-0.5%, a concentration of 0.08% being generally suitable), a magnesium salt (e. g. magnesium sulphate heptahydrate at 0.05% or thereabouts, which also conveniently adds sulphate which is also necessary but which may be added in some other form if another salt of magnesium is used), a phosphate (e. g. potassium dihydrogen phosphate at ODS-0.5%, 0.1% being a suitable concentration), a potassium salt (which may be conveniently the phosphate, see above) and traces (ca. [-10 p. p. m. of metal) of salts of iron, copper, zinc, manganese and a trace (ll p. p. m. of metal) of a molybdate.

Fermentations can be conveniently carried out at a temperature in the range 2533 C.

Not all strains of the fungus produce gibberellic acid. Results of tests carried out on a variety of strains do indicate however that strains isolated from rice seedlings infected with the bakanae" disease are more likely to produce gibbcrellic acid than strains isolated from other host plants such as cotton, maize and pineapple. (TI-f strains found on sugar cane some are active in PlCCillLitit'. gibberellic acid, others are not. Strains of the it which produce the acid are morphologically indistinguislr able from strains which produce none, and whether or not a strain is active can be determined only by growing it and testing the culture.

Strains of Gihberella fujikuroi which do produce gibbcrellic acid are the so-called Sawada and MW-alto" strains obtainable from the Centraalbureau voor Schiznmclcuiturcs at Baarn, Holland. Four strains numbered 1001, i004, 1135 and 1139 in our collection of moulds are also productive of gibberellic acid. Samples of these strains have been deposited with the Centraalbureau and with the Commonwealth Mycological Institute, Kew. Surrey. Cultures of these strains when grown as described herein will contain gibberellic acid.

A method of obtaining gibberellic acid comprises generally the following steps:

(i) Growth of a mould culture in an aqueous medium;

(ii) Filtration and extraction of the medium with an adsorbent;

(ii) Elution of the adsorbent;

(iv) Purification of the acid obtained by the elution.

Gibberellic acid has been produced in still and in stirred culture using Raulin-Thom medium containing sucrose as the carbon source.

EXA RIFLE I For still culture the Raulin-Thorn medium was dispensed in one litre portions in earthenware vessels and the cultures incubated for about ten days at 25. Incubation of stirred cultures lasted three to five days at 25. The culture filtrate. of pH 4 or less (adjusted with N- hydrochloric acid if necessary), was extracted with g./l. of B. D. H. activated charcoal. The air dried charcoal (containing to moisture) was continuously eluted with acetone. and the eluate evaporated under reduced pressure to give an aqueous concentrate which was ex tracted twice with at least an equal volume of ethyl acetate. The combined ethyl acetate solutions were cX-- tracted twice with 25% of their volume of phosphate buffer pH 6.3 (136 g. KH PO and 24 g. KOH in 1 l. of water). The buffer extracts were combined, adjusted to pH 3.8, and extracted twice with an equal volume of ethyl acetate. On concentrating these extracts, crude gibberellic acid crystallised in yields of about mg./1. The crude acid was purified by adding an equal volume of light petroleum (60-80" C.) to a solution in boiling ethyl acetate. Repetition of this process gave pure gibbcrellic acid as jagged lathes decomposing at 233-235 C.. optical rotation [111 +86 (c., 2.12 in ethanol). (Found: C, 65.9. 65.85: H. 6.6, 6.55. Eq. wt., 342. C H O re quires C, 65.9; H, 6.4% eq. wt. (monobasic) 346.) The molecular weight has been confirmed by the X-ray method. The unit cell was tetragonal and had a=b:10.905-. 0:28.74 A. The density was 1.34:0.01, whence the molecular weight (8 molecules per unit cell) i 345.

Direct extraction of the culture filtrate with organic 4 solvent will remove gibberellic acid but not so effectively as when charcoal is used as in the above Example I.

The empirical formula for the acid and derivatives would be COOX [ClB HIOI1OY OY where X may be hydrogen, alkyl or aryl radical or metal and i may be hydrogen or acyl radical.

Uibbcrellic acid gives a methyl ester, M. P. 209-210 C, optical rotation [nail +75 (c., 0.5 in ethanol) (found: C, 66.65; H, 6.85; OMe, 8.5. C H O requires -27. 66.65; H, 6.7; OMe, 8.6%), with either diazomethane or methyl iodide and potasium carbonate in acetone. The p-bromophenacyl ester has M. P. 218219 C. (found: C, H, Br, C27H27O7BI'. H20 requires C, 57.75; H, 5.2; Er, 14.2 Acetylgibberellic acid, M. P. 233-234" C. (deo) optical rotation [u] +152 (o, 0.5 in ethanol) (found: C, 65.1; H, 6.35; C H O requires C. 64.9; H, 6.2%), prepared with acetic anhydride in pyridine at room temperature, yields a methyl ester. M. P. l181 C. optical rotation [111 (c.. 0.4 in ethanol) (found: C, 65.3; H, 6.7. C I-1 0 requires C, 65.65; H, 6.5%), also obtained by acetylating methyl gibberellate.

As further examples of alkyl esters of gibberellic acid the following were prepared in each case by interaction of the earboxyl group of gibberellic acid and the appropriate diazohydrocarbon in a suitable solvent: ethyl gibberellate forms a hemi-methanolate, prismatic needles. M. P. 150-151 C. (found: C, 66.6; H, 7.6. C H O V2CH O requires C, 66.2; H, 7.2%) which on being heated to 140 C. in vacuo yields the solvent-free ester, M. P. -156 C. (found: C, 67.3; H, 7.2. C l-1 0,, requires C, 67.4; H, 7.0% Isopropyl gibberellate forms a hemi-rnethanolate, needles, M. P. 151-152 C. (found: C, 66.5; H, 7.7. C H O /zCH O requires C, 66.8; H, 7.5%). Butyl gibberellate hemi-methanolate forms prismatic needles, M. P. lS2l53 C. (found: C, 67.6; H, 7.7. C H O /2CH,,O requires C, 67.4; H, 7.7%). Octyl gibberellate forms prismatic needles, M. P. 157 159 C. (found: C, 70.2; H, 8.3. (1 4 0,; requires C, 70.7; H, 8.4%

Further examples of esters formed by acylation of an hydroxyl group in gibberellic acid are: butyryl gibberellic acid, M. P. 192 C. (found: C, 66.1; H, 7.0. C H O requires C, 66.2; H. 6.8%) prepared with butyric anhyddride in pyridine at room temperature, and benzoyl gibberellic acid, M. P. 204205 C. (found: C, 69.2; H, 5.9. tJ H O- requires C. 69.3; H, 5.8%) prepared with benmyl chloride in pyridine at room temperature.

Examples of derivatives of gibberellic acid in which the carboxyl group is esterified and an hydroxyl group is acylated are as follows: ethyl acetyl gibberellatc, M. P. 202204 C. (found: C, 66.0; H, 6.9. CzgHzgOq requires C, 66.3; H, 6.8%), isopropyl acetylgibberellate. M. P. 202-203 C. (found: C, 66.7; H, 7.1. C H O requires C, 67.0; H, 7.0%), butyl acctylgibberellate, M. P. 147 149 C. (found: C. 67.1; H, 7.2. C H O requires C. 67.5; H, 7.3%), ethyl butyrylgibberellate, M. P. -176 C. (found: C, 67.2; H, 7.5. C H O requires C. 67.5; H, 7.3%) and ethyl benzoylgibberellate, M. P. 192 C. (found: C. 70.5; H. 6.5. C23H30O7 requires C, 70.3; H. 6.3%), and octyl acetylgibberellate which consist of a colourless syrup, B. P. 195 202 C. (bath temperature)! 5 10 mm. (found: C, 69.6; H, 8.0. CggHwOq requires C, 69.6; H, 8.1%).

Diacetylgibberellic acid prepared by allowing gibberellic acid to react with acetic anhydride in pyridine at room temperature for 5 days formed prisms, M. P. 186.5487 C. (found: C, 64.25; H, 6.15. C I-I O requires C, 64.2; H, 6.1%). Methyl diacetylgibberellate was prepared by the action of excess ethereal diazomethane on a methanolic solution of diacetylgibberellic acid. It crystallised from methylene chloride-light petroleum (B. P. 60-80 C.) and from acetone-light petroleum (B. P. 60-80 C.) in massive prisms, M. P. 166.5-168.5 C. (found: C, 65.1; H, 6.45. C JI O requires C, 64.85; H, 6.35%).

An example of the procedure utilized to prepare the methyl ester of gibberellic acid is shown in the following example.

EXAMPLE II (A) A solution of 207 mg. of gibberellic acid in 7 ml. of methanol was cooled to 0 C. and an excess of diazomethane in ether was added with stirring. Five hours after the completion of the addition, the solution was placed on a water-bath and rapidly evaporated to dryness. The residue was crystallised from a benzene-methanol mixture to give 159 mg. of methyl gibberellate melting at 208-210" C. After a second crystallisation, the ester melted at 209-2l0 C. and had an optical rotation of [M +75, when determined in a 0.5 percent solution in ethanol.

(B) A mixture of 125 mg. of gibberellic acid, 0.3 ml. of methyl iodide, 0.7 g. of anhydrous potassium carbonate and 7 ml. of dry acetone was heated under reflux for 8 hours. washed with water and dried. Repeated crystallisation from a mixture of ethyl acetate and benzene gave methyl gibberellate melting at 204205 C.

Of coure, the other esters, as generally noted heretofore, can similarly be prepared.

The preparation of an ester from reaction of the gibberellic acid with an acyl-group-containing compound is illustrated by the following example, which shows the general method of preparation of such esters.

EXAMPLE In To a solution of 113 mg. of gibberellic acid in 1.8 ml. of pure dry pyridine was added 1 ml. of acetic anhydride. The mixture was left for 45 hours at room temperature and then the solution was placed on a water-bath and the solvents were rapidly distilled off in vacuo. The residue was dissolved in sodium bicarbonate solution, the solution filtered, and the filtrate acidified with hydrochloric acid. he resulting precipitate was collected by filtration and crystallised from a mixture of ethyl acetate and light petroleum (B. P. 60*80" C.) giving acetylgibherellic acid as rhombs meiting at 233-234 C. (decomposition) and having an optical rotation of [(11 +152", when determined in a 0.5 percent solution in ethanol.

It should be stressed that other esters of. gibberellic acid are within the scope of this invention, as, for example, the glycol and phenyl esters. The foregoing are merely by way of illustration.

In addition, it should be noted that various salts of gibberellic acid have been prepared. Particularly the alkali metal and alkaline earth salts are of interest, as for example: sodium gibberellate sesquihydrate, indefinite M. P. at 230238 C. (decomposition) (found: C, 57.4; H, 6.15. 2C H O Na, 3H O requires C, 57.7; H, 6.12%), potassium gibberellate sesquihydrate, indefinite M. P. at 225229 C. (decomposition) (found: C, 55.6; H, 5.7. 2C H O K, 3H O requires C, 55.5; H, 5.9%) and calcium gibberellate trihydrate, decomposition at 230-270 C. (found: C, 58.7; H, 6.0. (C19H21O5)gCa, 3H O requires C, 58.2; H, 6.2%).

The following example illustrates a method for preparing the sodium salt of gibberellic acid. It can be appreciated that the other salts generally noted heretofore can be similarly prepared.

EXAMPLE IV A solution of gibberellic acid (94.03 mg.) in methanol which had been freshly distilled over sodium hydroxide (3 ml.) was treated dropwise with the calculated volume The acetone was distilled off and the residue (10.93 ml.) of approximately 0.02 N carbonate-free, aqueous sodium hydroxide, the volume being calculated on the basis of a parallel titration, to phenolphthalein, of an aliquot portion (58.20 mg.) of gibberellic acid which required 6.76 ml. of the alkali solution for neutralization. The first mentioned solution of sodium gibberellate was rapidly evaporated under reduced pressure at room temperature. The residual sodium salt was dried for 7 days in a vacuum desiccator over phosphorus pentoxide; it consisted of transparent flakes, which disintegrate at ca. 160" C. to a white powder becoming yellow-brown at 210-215 C. and melting at 230-238" C. with decomposition, of sodium gibberellate sesquihydrate (found: C, 57.4; H, 6.15. 2C1H2108Na, 3H O requires C, 57.7; H, 6.12%).

To obtain maximum yields in stirred cultures it is necessary to carry fermentation on for what is a long time by usual standards. This is shown by the following yield/ time data for litre batch of Raulin-Thom medium containing 4% sucrose, initially adjusted to pH 3.5 and at 25 C.

TABLE II Hrs. fermentation Yield (mg/l.)

The time at which gibberellic acid begins to accumulate in a culture of a suitable strain of Gibberella fujikuroi and the final yield of gibberellic acid are influenced by the concentrations of the carbon source and nitrogen source and by the ratio of these two concentrations in the medium used.

The following examples are given in illustration.

A gibberellic acid producing strain of the mould was grown in 30 litre batches of a medium of the following composition:

The medium was stirred, aerated at a rate of 10 litres/ minute and maintained at a temperature of 25 C. The pH value of the medium was adjusted to 4.5 before sterilizing.

Yields of gibberellic acid (mg/1.) at various times during fermentation on media containing different concentrations of glucose and ammonium nitrate are given in the following tables.

4 TABLE III Yield of gibberellic acid (mg/I.)

Hours of Concentration of NHtNO: (ti-[ Glucose Monohydrate tirrtneua on These results show (a) the yield of gibberellic acid increases with increasing ratio of glucose/NH NO (b) the yield of gibberellic acid increases with increasing glucose;

TABLE IV Yield of gibberellic acid (mg/i.)

Percent Music, 0.90 0.48 l 0.24 0.12 Percent Dextrose: I

is. 94 211 l 310 14s a no l 135 200 13 54 11s 5 5 i s 50 (c) in media of given glucose concentration production of gibberellic acid commences soonest in media of low NH NO concentration; (d) in media of given NH IWJ concentration production of gibberellic acid commences soonest in media of low glucose concentration: (a) gibberellic acid is produced by the fungus only during times when it is accumulating or subsisting upon internal reserves of fat and carbohydrate, that is to say when active growth of the fungus has been checked by exhaustion of either the nitrogen or carbon in the nutrient. The best conditions for production of the acid appear to be those in which nitrogen is the factor which limits active growth and carbon is in excess though, of course, it is necessary that suliicient nitrogen be originally present in the nutrient to permit adequate growth of the fungus before acid production commences.

in general. high yields of gibberellic acid are obtained by growing the mould in a nutrient containing from 02-03% ammonium nitrate (or equivalent source of nitrogen) and from -30% glucose (or equivalent source of carbon) i. e.. from (LOT-0.1% N and from 3.540% C.

Gibberellic acid, and certain of its derivatives such P; salts and esters particularly acetyl gibberellic acid, diacetyl gibberellic acid, butyryl gibberellic acid and monohcnzoyl gibberellic acid have the property of promoting the growth of plants. as is shown by the following description of experiments on wheat and pea seedlings and grass.

When wheat seedlings are grown in water culture and gibberellic acid is added to the culture solution in proportions of from l-l0 parts per million of solution, the

seedlings grow taller than untreated plants. Increases in height of the order of 50% are common, the increase being due to (a) a great increase in length of the meso cotyl and (b) a smaller increase in the length of later internodes and of leaves. The leaves, although larger, are narrower than those of untreated plants. The enlarged plants are paler in colour than untreated plants in solutions of low nutrient status but this effect can be offset by increasing the concentration of nutrients in the culture solution.

Results from four experiments, where gibberellic acid was supplied in the culture solution at 5 p. p. m.. are sum marised in Table V. It will be seen that (a) fresh weight and dry weight of shoots are fairly consistently increased; (bl fresh weight and dry weight of roots are reduced. particularly in the low-level nutrient. The original data from which Table V was compiled also showed the. the weight increases in the sheet are usually far greater than the losses in weight of the roots, so that the net ril'cct of gibberellic acid on the whole plant is to increase fresh weight and dry weight. if the shoots alone are considered, the dry weight increases are due partly to redistrihution of dry matter between root and shoot. and partly due to greater dry matter synthesis by the plantv The carbon, nitrogen, phosphorus and ash contents of equal weights of material from treated and untreated plants do not dilfer greatly, though there appears possibly to he an increase in the percentage carbon content and a slight fall in ash, nitrogen and phosphorus. In any case, there is an undoubted great increase in the carbon assimilated per plant. This is a point of some interest; it could be due to the treated plants having a greater leaf area in which photosynthesis could take place or it could be due to an increase in the rate of carbon assimilation per unit area of leaf. In experiments where the necessary estimates of leaf area have been made it appears that the increased carbon assimilation is due to increased leaf area. There are qualitative differences in the carbon components of the treated plants. For example the total carbohydrate soluble in ethanol is increased by about 75% over controls, and by chromatography of sap expressed from cytolysed plants, it has been found that the sucrose content is increased by about 25??- and Fructose and glucose concentration by about lOOWw. it appears therefore that, as a result of the treatment, glucose replaces sucrose as the main sugar constituent of the p nuts.

TABLE V llhear: nzean percentage increase or decrease per seedling after 3 weeks growth resr f::"ag from ubbers Hie acid treatment "Statistically slgnitlhiutt (1 =01 r Statistically significant (P=.06Il.

not statistically analysed.

P W=iresh weight.

DW=vlry weight The eiTect of gibbcrellic acid on next smiling-1 growinr in water culture is even more striking than the effect on wheat seedlings. The height of the plant is greatly in creased-in some experiments the seedlings produced have been five times the height oi trol plan r 15 due to a great increase in the lcizth oi" lilia'l'lll i tfr The plants are much paler in colour t In; control plants.

The effects on the chemical comptmition of pea ECGLilings is similar to the eifects on wheat seedlings. TilCi'L is a great increase in fresh weight of shoots and a smaller,

but still considerable increase in dry weight. Fresh weight and dry weight of roots falls as a result of gibberellic acid treatment. Th .318 is an increase in the amount of carbon, nitrogen, ash, phosphorus and potassium per plant in the shoots, though in terms of percentage of dry weight the content of all these except carbon fell slightly. In the roots all these fall in amount.

The amount of carbohydrate soluble in 80% ethanol increased, as a result of gibberellic acid treatment, by about 30% taking the plant as a whole, by 40% if the shoots only were considered. As in the case of wheat, the glucose content of expressed sap was markedly in creased.

Reduced root growth is not necessarily obtained under all conditions. For example When gibberellic acid is applied to pea shoots, in a lanolin paste, increased weight of both roots and shoots is obtained.

The eifect on pea seedlings of some derivatives of gibberellic acid is shown in Table VI.

TABLE VI Oompou nd A pplied through Applied through roots lenvcs Acetyl glbherellic acid Diacetyl glbbercllic acid. Methyl acetyl gibb rcll ite. Monobcnzoyl gibberellic acid. Ethyl gllab rvll'ite Butyl gliibcl'ellu. Butyl acctylgibb iso-propy] acct late.

iso-propyl glbborellie! a. Butyryl gibberellic neid Octyl gibberell ite. Ethyl acetrlcibberell Phcnyl ac tylclbh rcllntc. Sodium gibbcrellste scsqulhigh activity (equal to GA).

high activity moderate activity..-

high activity moderate activity. do

. high activity moderate activity...

high activity r do high activity (equal to GA). high activity.

slight activity. high activity (equal to A).

Do. high activity (equal to GA).

inactive.

. not tested.

high activity (equal to GA).

hydrate.

Potassium gibbercllate seshigh activity (equal Do.

quihydrate. to GA).

Calcium gibhcrcilato ses- .do. Do.

quihydratc.

Applied through roots in culture solution containing the compound at 9. concentration of if) p. p. in.

Applied through leaves in doses of iOyjplaut in .002 ml. alcohol.

GA=gibhcrcllic acid.

The degree of activity of gibberellic acid derivatives appears to fall off with increasing substitution. For example, when gibberellic acid is esterified with higher alcohols the degree of activity falls off as may be seen in Table VI, though the octyl ester still retains some activity. On the other hand, although acetyl gibberellic acid is as active as gibberellic acid itself, as this substituted acid is esterified with alcohols of higher molecular weight so the activity of the ester falls off, as will be noted in Table VI, until it is negligible, in this particular case, in octyl acetyl gibberellate. Again, although diacetyl gibberellic acid is quite active, the methyl ester is substantially inactive.

Where a derivative is active when applied through the roots but not when applied through the leaves this may in fact be due to hydrolysis of the derivative in the soil yielding free gibberellic acid.

The fact that great increases in yield of dry matter may be obtained by treatment of wheat and pea seedlings with very small quantities of gibberellic acid is of considerable practical importance. Experiments in which grass crops were treated with gibberellic acid have confirmed that similar increases in dry matter yield can be obtained under field conditions.

In a pasture trial it was found that applications of gibbereilic acid (1-4 oz./acre) as an aqueous spray brought about an increase in the dry matter yield of herbage at the first cut. (See following Table VII.) 0, l, 2, 3 and 4 oz. gibberellic acid /acre were applied along with O, 3 and 6 cwts. fertiliser* per acre to ,6 acre *The fertiliser was one containing 12% N, 12% total Plol (11.9% water soluble) and 15% KrO.

plots on April 13, 1954, and the plots harvested on May 11, 1954.

TABLE VII Mean dry matter yields of herbage (cwts./acre) Gibberellic A cid (ozJacre) Fertiliser tewtJacre) 0 l 2 ll 4 Although substantial increases in dry matter yield were obtained without application of fertiliser, the grass plants were yellow and mechanically weak. However the plants on the gibberellic acid plus fertiliser (6 cwt./acre) plots were normal in appearance.

In general, it has been found that, in order to obtain the most beneficial results from the growth-promoting properties of gibberellic acid, treatment with the acid should be accompanied by treatment with additional quantities of plant nutrients, particularly nitrogen, which may be derived from a mixed fertilizer as above or from a source of nitrogen such as urea, sulphate of ammonia and nitrate of ammonia. A suitable rate of application of nitrogen which will substantially reduce or prevent the yellowing of the foliage on stimulation of growth is from 40-80 lb. N/acre.

One of the most important aspects of the present invention in relation to increased production of grass is that applications of gibberellic acid or an active derivative to grass land in the early spring, autumn or winter will cause the grass to grow under conditions of low light intensity and low temperature, i. e., conditions under which grass could not be stimulated into growth merely by application of a fertiliser. As a result, it is possible to extend the grass growing season and so obtain an earlier bite for livestock or an earlier cut for hay or silage. The simultaneous application of fertiliser leads of course to earlier use of the fertiliser in the earlier growth.

The gibberellic acid or an active derivative thereof may be applied in a dry form, i. e., as a powder, in admixture with a diluent which may be inert, e. g., china clay, fuliers earth, kieselguhr or lime, or which may contain a plant nutrient, e. g. mixed fertiliser or sulphate or nitrate of ammonia. Alternatively it may be applied as a spray, the active constituent then being dispersed in water. The aqueous dispersion may additionally contain plant on trients such as urea or an ammonium salt.

Preferably the active constituent is so applied that its main effect is achieved by absorption through the fo age rather than through the roots since in the latter ciri 1 stances it may depress the rate of root growth. Proton ably therefore it is applied as a spray, more preferab! a low volume spray, say at a rate of 10 or 20 gallons of spray to the acre to give a distribution of 2 ozjncre of gibberellic acid or equivalent of active derivative.

A convenient method of preparing an aqueous dispersion of gibberellic acid or an active derivative is tr.- dissolve the acid or derivative in a water-miscible solvent and pour the solution with stirring into water.

Although we have described processes for isolating pure gibberellic acid from cultures of Gibberella fujikuroi, the pure material being required for chemical analysis and general identification of the acid, it will be obvious that for agricultural purposes it will not be necessary to isolate the pure acid, a crude concentrate being adequate for treatment of plants or of ground in which plants are growing or are to grow.

We claim:

1. A method for promoting the growth of plants comprising treating said plants with a gibberellic acid compound having the formula said acid being characterized by the following properties:

Molecular formula, C H O Melting point, 233-235 C. (vigorous gas evolution) Optical rotation, [111 +83 (c., 0.51 in methanol) Monomethyl ester:

Melting point, 209210 C.

Optical rotation, [a1 +75 (c., 0.5 in ethanol) p-Bromophenacyl ester:

Melting point, 218-219 C.

Hydrolysis with boiling mineral acid-1 mole carbon dioxide evolved Acetyl derivative:

Melting point, 233-234 C. (dec.)

Optical rotation, [al +152 (c., 0.5 in ethanol) Methyl acetyl gibberellatc:

Melting point, 180-181 C.

Optical rotation, [ah-) +150 (c., 0.4 in ethanol) Absorption bands in the infra-red region of the spectrum when suspended in the form of a Nujol mull at the following frequencies expressed in reciprocal centimeters:

Absorption Absorption 55% and 45%-54% above and wherein X is selected from the group consisting of hydrogen, alkali metal, alkaline earth metal, lower alkyl, phenyl, hydroxyethyl, and para-bromo-phenacyl, and wherein Y and Z are each selected from the group consisting of hydrogen, and

O n-iiwhere R is selected from the group consisting of lower alkvl, and phenyl.

2. The method of claim 1 in which the compound is dispersed in water as diluent and is applied as a spray.

3. The method of producing gibberellic acid, said acid being characterized by the following properties:

12 Absorption bands in the infra-red region of the spectrum when suspended in the form of a Nujol mull at the following frequencies expressed in reciprocal centimeters:

Absorption A bsorptlon 55% and 45%-54% above 3, 390 1,328 3, 460 1, 263 1, 752 1. 250 1, 191 1 156 l, 172 l. 128 1,032 1, 1. 021 978 839 l 778 comprising cultivating Gibberella fufikuroi in an aqueous medium including a carbon and nitrogen source wherein said carbon is in excess, and continuing cultivation after active growth has ceased.

4. A method for producing gibbercllic acid, said acid being characterized by the following properties:

Molecular formula, C H O Melting point, 233-235 C. (vigorous gas evolution) Optical rotation, [01 +83 (c., 0.51 in methanol) Monomethyl ester:

Melting point, 209-210 C.

Optical rotation, [al +75 (c., 0.5 in ethanol) p-Bromophenacyl ester:

Melting point, 218-219 C.

Hydrolysis with boiling mineral acid-1 mole carbon dioxide evolved Acetyl derivative:

Melting point, 233-234 C. (dec.)

Optical rotation, [al +152 (c., 0.5 in ethanol) Methyl acetyl gibberellate:

Melting point, ISO-181 C.

Optical rotation, [tfl (c., 0.4 in ethanol) Absorption bands in the infra-red region of the spectrum when suspended in the form of a NujoP mull at the following frequencies expressed in reciprocal centimeters:

Absorption Absorption 55% and V -54% above comprising cultivating Gibberella fujikuroi in an aqueous medium including a carbon and nitrogen source wherein said carbon is in excess and in amount equivalent to 240% w./v. of glucose and the nitrogen is in amount of 0.01-0.5%, and continuing the cultivation after active growth has ceased.

5. The method for producing gihberellic acid, said acid being characterized by the following properties:

Molecular formula, C H O Melting point, 233-235 C. (vigorous gas evolution) Optical rotation, [c1 +83 (c., 0.51 in methanol) Monornethyl ester:

Melting point, 2092l0 C.

Optical rotation, EaJ +75 (c., 0.5 in ethanol) Absorption bands in the infra-red region of the spectrum when suspended in the form of a Nujol mull at the following frequencies expressed in reciprocal centimeters:

Absorption A bsorption 55% and 45%-54% above comprising cultivating Gibberella fujikuroi in an aqueous medium including a carbon and nitrogen source wherein said carbon is in excess in amount equivalent to 10-30% glucose and the nitrogen is in amount of 0.07-0.l%, the carbon source being selected from the group consisting of glucose, sucrose and glycerine, and the nitrogen source being selected from the group consisting of an ammonium salt, a nitrate, and a protein digest, and continuing the cultivation after active growth has ceased, filtering the resulting culture, extracting said culture filtrate with activated charcoal, continuously eluting the charcoal containing the extract with acetone, evaporating the eluate under reduced pressure to give an aqueous concentrate, extracting said concentrate with ethyl acetate, extracting the resulting ethyl acetate solution with a phosphate buffer (KH PO and KOH), extracting the resulting bufifer extract with ethyl acetate, concentrating the resulting extract to obtain crude gibbereilic acid crystals, purifying said crystals by adding light petroleum to a solution thereof in boiling ethyl acetate and thereafter separating the pure gibbereilic acid.

6. Gibbereilic acid compounds having the formula C X [CMHio JOY OZ said acid being characterized by the following properties:

Molecular formula, C H O Melting point, 233-235 C. (vigorous gas evolution) Optical rotation, [aJ +83 (c., 0.51 in methanol) Monomethyl ester:

Melting point, 209-210 C.

Optical rotation, [ai +75 (c., 0.5 in ethanol) p-Bromophenacyl ester:

Melting point, 218-219 C.

Hydrolysis with boiling mineral acid-l mole carbon dioxide evolved Acetyl derivative:

Melting point, 233-234 C. (dec.)

Optical rotation, lai +152 (c., 0.5 in ethanol) Methyl acetyl gibberellate:

Melting point, l80-181 C.

Optical rotation, [ai +150 (c., 0.4 in ethanol) Absorption bands in the infra-red region of the spectrum when suspended in the form of a Nujol mull at the following frequencies expressed in reciprocal centimeters:

Absorption Absorption 55% and 45%-54% above and wherein X is selected from the group consisting of hydrogen, alkali metal, alkaline earth metal, lower alkyl, phenyi, hydroxyethyl, and para-bromo-phenacyl, and wherein Y and Z are each selected from the group consisting of hydrogen, and

i R i where R is selected from the group consisting of lower alkyl, and phenyl.

7. A plant nutrient comprising a gibberellic acid compound having the formula COOK [CrsHmOzJOY 0 Z and a nitrogen containing fertilizer in which said compound is uniformly distributed, said acid being characterized by the following properties:

Absorption bands in the infra-red region of the spectrum when suspended in the form of a Nujol" mull at the following frequencies expressed in reciprocal centimeters:

Absorption Absorption 55% and 45%-64% above and wherein X is selected from the group consisting of hydrogen, alkali metal, alkaline earth metal, lower alkyl, phenyl, hydroxy-ethyl, and para-bromo-phenacyl, and wherein Y and Z are each selected from the group consisting of hydrogen, and

all

15 where R is selected from the group consisting of lower alkyl, and phenyl.

8. A gibberellic acid compound as claimed in claim 6 wherein said compound is the alkali metal salt of gibberellic acid.

9. A gibberellic acid compound as claimed in claim 6 wherein said compound is the alkaline earth metal salt of gibberellic acid.

10. A gibberellic acid compound as claimed in claim 6 wherein said compound is the sodium salt of gibberellic acid.

11. A gibberellic acid compound as claimed in claim 6 wherein said compound is the potassium salt of gibberellic acid.

Yabuta et al.: Chemical Abstracts, vol. 33, columns 8238(0) to 8239(a), 1939.

Yabuta et 211.: Chemical Abstracts, vol. 44, columns 10814(a) to 10817(c), 1950.

Sumiki: Chemical Abstracts, vol. 48, columns 12920(a) to 12921, 1954 (abstract of 1952 article).

Kato: Chemical Abstracts, vol 48, column 8886(i), 1954 (abstract of 1951 article).

Formula Index of Chemical Abstracts, vol. 47, page 264F, 1954 (refers to column 6442(i) and abstract of 1952 source).

Formula Index of Chemical Abstracts, vol. 48, page 2995, 1953 (refers to column 5204(d)).

Gibberellins for Growth, Chem. and Eng. News, pages 4496 and 4501 (Sept. 17, 1957).

Report of the Chief of the Bureau of Agr. and Ind. Chem, U. S. Dept. of Agr. Res. Adm., 1953, page 74.

Stodola et al.: Arch. Biochem, vol. 54, pages 240" 245 (1955).

Cross: J. Chem. Soc, 1954, pages 4670-76. 

1. A METHOD FOR PROMOTING THE GROWTH OF PLANTS COMPRISING TREATING SAID PLANTS WITH A GIBBERELILIC ACID COMPOUND HAVING THE FORMULA 